Acetoacetic acid oxidation

Recently extracts of acetone powder and homogenates of liver have been obtained which specifically oxidize tyrosine, p-hydroxyphenyl-pyruvic acid, and homogentisic acid to acetoacetic acid. The reaction is aerobic and requires the uptake of 4 atoms of oxygen per molecule for the oxidation of either L-tyrosine or p-hydroxyphenylpyruvic acid to yield 1 molecule of CO2 and 1 of acetoacetic acid oxidation of homogentisic acid to acetoacetate requires the uptake of 2 atoms of oxygen. 

Dihydroxyphenylpyruvate was not oxidized to homogentisate, thus it cannot be an intermediate in the oxidation of p-hydroxyphenyl-pyruvate. The results suggest that the hydroxylation shift of the side chain and decarboxylation of the p-hydroxyphenylpyruvate are simultaneous processes. Additional evidence that 2,5-dihydroxyphenylpyruvate is not the intermediate was obtained by experiments in which the relative rates of oxidation of this compound and of p-hydroxyphenylpyruvate were compared in homogenates of rat liver, where the reaction proceeded to formation of acetoacetic acid. Oxidation of p-hydroxyphenylpyruvate proceeded much more rapidly. Other analogs of p-hydroxyphenylpyruvate were found to be inactive as substrates. 

Fig. 6. Key intermediates derived from benzene. The alkylation reaction shown employs ethylene oxide. Hydrazine condenses with acetoacetic acid to form...

The rate of mitochondrial oxidations and ATP synthesis is continually adjusted to the needs of the cell (see reviews by Brand and Murphy 1987 Brown, 1992). Physical activity and the nutritional and endocrine states determine which substrates are oxidized by skeletal muscle. Insulin increases the utilization of glucose by promoting its uptake by muscle and by decreasing the availability of free long-chain fatty acids, and of acetoacetate and 3-hydroxybutyrate formed by fatty acid oxidation in the liver, secondary to decreased lipolysis in adipose tissue. Product inhibition of pyruvate dehydrogenase by NADH and acetyl-CoA formed by fatty acid oxidation decreases glucose oxidation in muscle. 

Under metabolic conditions associated with a high rate of fatty acid oxidation, the liver produces considerable quantities of acetoacetate and d(—)-3-liydroxyl)utyrate (P-hydroxybutyrate). Acetoacetate continually undergoes spontaneous decarboxylation to yield acetone. These three substances are collectively known as the ketone bodies (also called acetone bodies or [incorrectly ] ketones ) (Figure 22-5). Acetoacetate and 3-hydroxybu-... 

The ketone bodies (acetoacetate, 3-hydroxybutyrate, and acetone) are formed in hepatic mitochondria when there is a high rate of fatty acid oxidation. The pathway of ketogenesis involves synthesis and breakdown of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by two key enzymes, HMG-CoA synthase and HMG-GoA lyase. 

A number of 2H-1,2,3-triazole 1-oxides 72 were prepared by chemists at the Cassella Company as potential NO-donors in view of their formal structural similarity with furoxan derivatives [18]. Derivative 72a was studied in depth. It was obtained by cupric sulfate oxidation of intermediate 79, derived from the action of the substituted phenylhydrazine 78 on the oximino acetoacetic acid amide 77 (Scheme 6.13). 

Perfusion experiments before oxygenation problems were solved often gave incomplete or misleading information. Embden s work on fatty acid oxidation in liver (see Chapter 7) suggested acetoacetate was a normal metabolite, although in the intact animal free acetoacetate is only detected when glucose oxidation is impaired. 

Between 1906 and 1908 the breakdown of fatty acids to acetone was detected by Embden in perfused livers. Only fatty acids with even numbers of carbon atoms produced this effect. The acetone was postulated to have originated from acetoacetate. For the next 30 years the 6-oxidative route of fatty acid oxidation was generally unchallenged. By 1935-1936 however much more accurate determinations of the yields of acetoacetate per mole of fatty acid consumed (Deuel et al., Jowett and Quastel) indicated convincingly that more than one mole of acetoacetate might be obtained from 6C or 8C fatty acids. (Octanoic acid was often used as a model fatty acid as it is the longest fatty acid which is sufficiently soluble in water at pH 7.0 for experimental purposes.) The possibility had therefore to be entertained that 2C fragments could recondense (MacKay et al. 1942). 

In some poorly controlled diabetic patients the high rate of fatty acid oxidation decreases the mitochondrial NADVNADH concentration ratio so that the 3-hydroxybutyrate/acetoacetate concentration ratio can rise to as high as 15 in the blood. Since a test for ketone bodies in the urine (using Clinistix or similar material) detects only acetoacetate this can result in a serious underestimate of the concentration of ketone bodies in the urine. 

T12. Fatty Acid Oxidation in Uncontrolled Diabetes When the acetyl-CoA produced during /3 oxidation in the liver exceeds the capacity of the citric acid cycle, the excess acetyl-CoA forms ketone bodies—acetone, acetoacetate, and D-/3-hydroxybutyrate. This occurs in severe, uncontrolled diabetes because the tissues cannot use glucose, they oxidize large amounts of fatty acids instead. Although acetyl-CoA is not toxic, the mitochondrion must divert the acetyl-CoA to ketone bodies. What problem would arise if acetyl-CoA were not converted to ketone bodies How does the diversion to ketone bodies solve the problem ... 

Synthesis of the ketone bodies HMG CoA is cleaved to produce acetoacetate and acetyl CoA, as shown in Figure 16.23, Acetoacetate can be reduced to form 3-hydroxybutyrate with NADH as the hydrogen donor. Acetoacetate can also spontaneously decarboxylate in the blood to form acetone—a volatile, biologically non-metabolized compound that can be released in the breath. [Note The equilibrium between acetoacetate and 3-hydroxybutyrate is determined by the NADVNADH ratio. Because this ratio is high during fatty acid oxidation, 3-hydroxy-butyrate synthesis is favored.]... 

Liver mitochondria can convert acetyl CoA derived from fatty acid oxidation into the ketone bodies, acetoacetate and (3-hydroxybutyrate. (Acetone, a nonmetabolizable ketone body, is produced spontaneously from acetoacetate in the blood.) Peripheral tissues possessing mitochondria can oxidize 3-hydroxybutyrate to acetoacetate, which can be reconverted to acetyl CoA, thus producing energy for the cell. 

Structures 64 and 65 were proposed in [7] for the product from the condensation of isatin 7 with acetoacetic acid (a P-keto acid). The first must clearly be preferred, since the CH2 group must be more active than CH3 under the conditions of the Pfitzinger reaction. Actually in [21] the structure of the product 64 was proved by its oxidation to the tricarboxylic acid 66, which was also synthesized from the keto dicarboxylic acid 67 and isatin 7. 

The first committed step in TA and nicotine biosynthesis is catalyzed by putrescine JV-methyltransferase (PMT) (Fig.7.4).82 A PMT cDNA isolated from tobacco showed extensive homology to spermidine synthase from mammalian and bacterial sources.83 A-Methylputrescine is oxidatively deaminated to 4-aminobutanal, which undergoes spontaneous cyclization to form the reactive A-methyl-A1-pyrrolinium cation. Although the enzymes involved are unknown, the A-methyl-A1-pyrrolinium cation is thought to condense either with acetoacetic acid to yield hygrine as a precursor to the tropane ring, or with nicotinic acid to form nicotine. 

Coupling, of benzenediazonium chloride with acetoacetic acid, 32, 84 of diazotized />-aminoacetophenone with quinone, 34, 1 of diazotized 3,5-dichloro-2-aminoben-zoic acid to give 4,4, 6,6 -tetra-chlorodiphenic acid, 31, 96 of diphenyldichloromethane to tetra-phenylethylene, 31,104 Creased flask, 37, 55 Creosol, 33,17 Crotonaldehyde, 33, 15 34, 29 diethyl acetal, 32, 5 Cupric acetate monohydrate, 36, 77 Cuprous oxide-silver oxide, 36, 36, 37 Cyanamide, 34, 67 36, 8 Cyanoacetamide, 32, 34 Cyanoacetic acid, 31, 25 38, 16, 18 Cyanoacetylurca, 37,16 /i-Cyanobenz d[Pg.99]

A pathway by which acetate and acetoacetate are oxidized is indicated in Fig. 8. The similarity to the mechanism of oxidation of pyruvate is striking. According to this proposed scheme acetate and acetoacetate are converted to a hypothetical two-carbon intermediate which is capable of condensing with oxalacetate or one of the other four-carbon dicarboxylic acids to yield cis-aconitate or isocitrate. The rest of the cycle is identical with the carbohydrate tricarboxylic cycle. Reference to this cycle illustrates several points of interest. It makes clear the pathway by which labelled carbon in acetate or acetoacetate may be transformed into D-glucose or glycogen. Since oxalacetate labelled in either carboxyl position is generated in the cycle, carboxyl-labelled... 

Acetyl-CoA is regenerated in this process. The overall product yields in moles per mole of glucose converted are approximately 0.5 acetate, 0.75 butyrate, 2 CO2, and 2 H2 2.5 mol ATP are formed. The nonacidic compounds, acetone, 1-butanol, and 2-propanol, are formed by transformation of some of the acetoacetyl-CoA into acetoacetic acid, which is the precursor of acetone and 2-propanol. Some of the butyryl-CoA is the precursor of 1-butanol via intermediate butyraldehyde. Ethanol is formed by reduction of small amounts of acetyl-CoA. The end result of the production of the neutral products by these additional pathways is that the yields of the other products are reduced. The neutral products are in a lower oxidation state than the acidic products and require additional reducing power as NADH to be formed. Some of the product Hj serves to sustain and provide NADH because higher partial pressures of H2 during the fermentation promote higher yields of the neutral products, whereas removal of the product H2 as it is formed has the opposite effect. 

Catabolism of tyrosine and tryptophan begins with oxygen-requiring steps. The tyrosine catabolic pathway, shown at the end of this chapter, results in the formation of fumaric acid and acetoacetic acid. Tryptophan catabolism commences with the reaction catalyzed by tryptophan-2,3-dioxygenase. This enzyme catalyzes conversion of the amino acid to N-formyl-kynurenine. The enzyme requires iron and copper and thus is a metalloenzyme. The final products of the pathway are acetoacetyl-CoA, acetyl-CoA, formic acid, four molecules of carbon dioxide, and two ammonium ions. One of the intermediates of tryptophan catabolism, a-amino-P-carboxymuconic-8-semialdehyde, can be diverted from complete oxidation, and used for the synthesis of NAD (see Niacin in Chapter 9). 

During prolonged starvation or when carbohydrate metabolism is severely impaired, as in uncontrolled diabetes mel-iitus (see Chapter 25), the formation of acetyl-CoA exceeds the supply of oxaioacetate. The abundance of acetyl-CoA results from excessive mobilization of fatty acids from adipose tissue and excessive degradation of the fatty acids by p-oxidation in the liver. The resulting acetyl-CoA excess is diverted to an alternative pathway in the mitochondria and forms acetoacetic acid, P-hydroxybutyric acid, and acetone—three compounds known collectively as ketone bodies (Figure 26-9). The presence of ketone bodies is a frequent finding in severe, uncontrolled diabetes melUtus. 

Acetoacetate and 6-hydroxybutyrate are products of normal metabolism of fatty acid oxidation and serve as metabolic fuels in extrahepatic tissues. Their level in blood depends on the rates of production and utilization. Oxidation increases as their plasma level increases. Some extra-hepatic tissues (e.g., muscle) oxidize them in preference to glucose and fatty acid. Normally, the serum concentration of ketone bodies is less than 0.3 mM/L. 

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